The actin filament is a key element in the dynamic machinery of motile cells and in the cytoskeleton of non-motile cells. Different assemblies of actin filaments fulfill different roles, even within the same cell. These assemblies differ both in their function and in their filament organization. We propose experiments to discover the properties of actin which make it a common element in the cytoskeletal structures. We also plan to study how these cytoskeletal structures are constructed. The approach, using combined techniques of cryo and conventional electron microscopy and image analysis, is to study the structure of isolated actin filaments, free and in combination with other proteins, actin bundles, and a cytoplasmic actin gel. In particular, we plan: 1. a study of actin and its auxiliary proteins in motile bundles obtained from the sperm of the horseshoe crab. The filaments obtained from this bundle are well ordered and therefore suitable for image analysis. The resulting three-dimensional maps will reveal the organization of the component proteins and will provide a map of the core actin filament. We will use the latter, together with the existing map of the subunit derived by X-ray analysis, to determine a near atomic model of the actin filament. 2. a study of the structure composition and assembly of the cuticular plate, an in vivo actin gel. This will be the first naturally occurring actin gel whose structure will be known in some detail. 3. a study of the structure of the actin-villin bundles. Villin is an actin-bundling and filament-severing protein. Images of the bundle in ice may reveal the structure of villin, how it interacts with actin and how it is able to sever filaments. 4. an in situ study of the actin cytoskeleton in yeast. Only three proteins and seven genes appear to be involved in the actin cytoskeleton. Because of its simplicity and genetic characterization, this system may be the ideal one in which to combine genetics, biochemistry and structural studies. The point of the proposed work is to see how tractable the system is for structural studies.